The present invention relates to inhibitors of the nuclear enzyme poly(adenosine 5xe2x80x2-diphospho-ribose) polymerase [xe2x80x9cpoly(ADP-ribose) polymerasexe2x80x9d or xe2x80x9cPARPxe2x80x9d, which is also referred to as ADPRT (NAD:protein (ADP-ribosyl transferase (polymersing)) and PARS (poly(ADP-ribose) synthetase) and provides compounds and compositions containing the disclosed compounds. Moreover, the present invention provides methods of using the disclosed PARP inhibitors to prevent and/or treat tissue damage resulting from cell damage or death due to necrosis or apoptosis; neural tissue damage resulting from, for example, ischemia and reperfusion injury, such as cerebral ischemic stroke, head trauma or spinal cord injury; neurological disorders and neurodegenerative diseases, such as, for example, Parkinson""s or Alzheimer""s diseases and multiple sclerosis; to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders, such as, for example, myocardial infarction; to treat other conditions and/or disorders such as, for example, age-related muscular degeneration, AIDS and other immune senescence diseases, inflammation, arthritis, gout, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, hyperglycemia, diabetes (such as diabetes mellitus), inflammatory bowel disorders (such as colitis and Crohn""s disease), acute pancreatitis, mucositis, hemorrhagic shock, splanchnic artery occlusion shock, multiple organ failure (such as involving any of the kidney, liver, renal, pulmonary, retinal, pancreatic and/or skeletal muscle systems), acute autoimmune thyroiditis, muscular dystrophy, osteoarthritis, osteoporosis, chronic and acute pain (such as neuropathic pain), renal failure, retinal ischemia, septic shock (such as endotoxic shock), local and/or remote endothelial cell dysfunction (such are recognized by endo-dependent relaxant responses and up-regulation of adhesion molecules), inflammation and skin aging; to extend the lifespan and proliferative capacity of cells, such as, for example, as general mediators in the generation of oxidants, proinflammatory mediators and/or cytokines, and general mediators of leukocyte infiltration, calcium ion overload, phospholipid peroxidation, impaired nitric oxide metabolism and/or reduced ATP production; to alter gene expression of senescent cells; or to radiosensitize hypoxic tumor cells.
PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose) synthetase), or ADPRT (for NAD:protein (ADP-ribosyl) transferase (polymerising)) is a major nuclear protein of 116 kDa. It is mainly present in almost all eukaryotes. The enzyme synthesizes poly(ADP-ribose), a branched polymer that can consist of over 200 ADP-ribose units from NAD. The protein acceptors of poly(ADP-ribose) are directly or indirectly involved in maintaining DNA integrity. They include histones, topoisomerases, DNA and RNA polymerases, DNA ligases, and Ca2+- and Mg2+-dependent endonucleases. PARP protein is expressed at a high level in many tissues, most notably in the immune system, heart, brain and germ-line cells. Under normal physiological conditions, there is minimal PARP activity. However, DNA damage causes an immediate activation of PARP by up to 500-fold. Among the many functions attributed to PARP is its major role in facilitating DNA repair by ADP-ribosylation and therefore co-ordinating a number of DNA repair proteins. As a result of PARP activation, NAD levels significantly decline. While many endogenous and exogenous agents have been shown to damage DNA and activate PARP, peroxynitrite, formed from a combination of nitric oxide (NO) and superoxide, appears to be a main perpetrator responsible for various reported disease conditions in vivo, e.g., during shock, stroke and inflammation
Extensive PARP activation leads to severe depletion of NAD in cells suffering from massive DNA damage. The short life of poly(ADP-ribose) (half-life less than 1 min) results in a rapid turnover rate. Once poly(ADP-ribose) is formed, it is quickly degraded by the constitutively active poly(ADP-ribose) glycohydrolase (PARG), together with phosphodiesterase and (ADP-ribose) protein lyase. PARP and PARG form a cycle that converts a large amount of NAD to ADP-ribose. In less than an hour, over-stimulation of PARP can cause a drop of NAD and ATP to less than 20% of the normal level. Such a scenario is especially detrimental during ischaemia when deprivation of oxygen has already drastically compromised cellular energy output. Subsequent free radical production during reperfusion is assumed to be a major cause of tissue damage. Part of the ATP drop, which is typical in many organs during ischaemia and reperfusion, could be linked to NAD depletion due to poly(ADP-ribose) turnover. Thus, PARP or PARG inhibition is expected to preserve the cellular energy level to potentiate the survival of ischaemic tissues after insult.
Poly(ADP-ribose) synthesis is also involved in the induced expression of a number of genes essential for inflammatory response. PARP inhibitors suppress production of inducible nitric oxide synthase (iNOS) in macrophages, P-type selectin and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells. Such activity underlies the strong anti-inflammation effects exhibited by PARP inhibitors. PARP inhibition is able to reduce necrosis by preventing translocation and infiltration of neutrophils to the injured tissues. (Zhang, J. xe2x80x9cPARP inhibition: a novel approach to treat ischaemia/reperfusion and inflammation-related injuriesxe2x80x9d, Chapter 10 in Emerging Drugs (1999) 4: 209-221 Ashley Publications Ltd., and references cited therein.)
PARP production is activated by damaged DNA fragments which, once activated, catalyzes the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones and PARP itself. During major cellular stresses the extensive activation of PARP can rapidly lead to cell damage or death through depletion of energy stores. As four molecules of ATP are consumed for every molecule of NAD (the source of ADP-ribose and PARP substrate) regenerated, NAD is depleted by massive PARP activation and, in the efforts to re-synthesize NAD, ATP may also be depleted.
It has been reported that PARP activation plays a key role in both NMDA- and NO-induced neurotoxicity. This has been demonstrated in cortical cultures and in hippocampal slices wherein prevention of toxicity is directly correlated to PARP inhibition potency (Zhang et al., xe2x80x9cNitric Oxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicityxe2x80x9d, Science, 263:687-89 (1994) and Wallis et al., xe2x80x9cNeuroprotection Against Nitric Oxide Injury with Inhibitors of ADP-Ribosylationxe2x80x9d, NeuroReport, 5:3, 245-48 (1993)). The potential role of PARP inhibitors in treating neurodegenerative diseases and head trauma has thus been recognized even if the exact mechanism of action has not yet been elucidated (Endres et al., xe2x80x9cIschemic Brain Injury is Mediated by the Activation of Poly(ADP-Ribose)Polymerasexe2x80x9d, J. Cereb. Blood Flow Metabol., 17:1143-51 (1997) and Wallis et al., xe2x80x9cTraumatic Neuroprotection with Inhibitors of Nitric Oxide and ADP-Ribosylationxe2x80x9d, Brain Res., 710:169-77 (1996)).
Similarly, it has been demonstrated that single injections of PARP inhibitors have reduced the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle in rabbits. In these studies, a single injection of 3-amino-benzamide (10 mg/kg), either one minute before occlusion or one minute before reperfusion, caused similar reductions in infarct size in the heart (32-42%) while 1,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reduced infarct size by a comparable degree (38-48%). Thiemermann et al., xe2x80x9cInhibition of the Activity of Poly(ADP Ribose) Synthetase Reduces Ischemia-Reperfusion Injury in the Heart and Skeletal Musclexe2x80x9d, Proc. Natl. Acad. Sci. USA, 94:679-83 (1997). These results make it reasonable to suspect that PARP inhibitors could salvage previously ischemic heart or skeletal muscle tissue.
PARP activation can also be used as a measure of damage following neurotoxic insults following over-exposure to any of glutamate (via NMDA receptor stimulation), reactive oxygen intermediates, amyloid xcex2-protein, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its active metabolite N-methyl-4-phenylpyridine (MPP+), which participate in pathological conditions such as stroke, Alzheimer""s disease and Parkinson""s disease. Zhang et al., xe2x80x9cPoly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damagexe2x80x9d, J. Neurochem., 65:3, 1411-14 (1995). Other studies have continued to explore the role of PARP activation in cerebellar granule cells in vitro and in MPTP neurotoxicity. Cosi et al., xe2x80x9cPoly(ADP-Ribose) Polymerase (PARP) Revisited. A New Role for an Old Enzyme: PARP Involvement in Neurodegeneration and PARP Inhibitors as Possible Neuroprotective Agentsxe2x80x9d, Ann. N. Y. Acad. Sci., 825:366-79 (1997); and Cosi et al., xe2x80x9cPoly(ADP-Ribose) Polymerase Inhibitors Protect Against MPTP-induced Depletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6 Micexe2x80x9d, Brain Res., 729:264-69 (1996). Excessive neural exposure to glutamate, which serves as the predominate central nervous system neurotransmitter and acts upon the N-methyl-D-aspartate (NMDA) receptors and other subtype receptors, most often occurs as a result of stroke or other neurodegenerative processes. Oxygen deprived neurons release glutamate in great quantities during ischemic brain insult such as during a stroke or heart attack. This excess release of glutamate in turn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, Kainate and MGR receptors, which open ion channels and permit uncontrolled ion flow (e.g., Ca2+ and Na+ into the cells and K+ out of the cel leading to overstimulation of the neurons. The over-stimulated neurons secrete more glutamate, creating a feedback loop or domino effect which ultimately results in cell damage or death via the production of proteases, lipases and free radicals. Excessive activation of glutamate receptors has been implicated in various neurological diseases and conditions including epilepsy, stroke, Alzheimer""s disease, Parkinson""s disease, Amyotrophic Lateral Sclerosis (ALS), Huntington""s disease, schizophrenia, chronic pain, ischemia and neuronal loss following hypoxia, hypoglycemia, ischemia, trauma, and nervous insult. Glutamate exposure and stimulation has also been implicated as a basis for compulsive disorders, particularly drug dependence. Evidence includes findings in many animal species, as well as in cerebral cortical cultures treated with glutamate or NMDA, that glutamate receptor antagonists (i.e., compounds which block glutamate from binding to or activating its receptor) block neural damage following vascular stroke. Dawson et al., xe2x80x9cProtection of the Brain from Ischemiaxe2x80x9d, Cerebrovascular Disease, 319-25 (H. Hunt Batjer ed., 1997). Attempts to prevent excitotoxicity by blocking NMDA, AMPA, Kainate and MGR receptors have proven difficult because each receptor has multiple sites to which glutamate may bind and hence finding an effective mix of antagonists or universal antagonist to prevent binding of glutamate to all of the receptor and allow testing of this theory, has been difficult. Moreover, many of the compositions that are effective in blocking the receptors are also toxic to animals. As such, there is presently no known effective treatment for glutamate abnormalities.
The stimulation of NMDA receptors by glutamate, for example, activates the enzyme neuronal nitric oxide synthase (nNOS), leading to the formation of nitric oxide (NO), which also mediates neurotoxicity. NMDA neurotoxicity may be prevented by treatment with nitric oxide synthase (NOS) inhibitors or through targeted genetic disruption of nNOS in vitro. Dawson et al., xe2x80x9cNitric Oxide Mediates Glutamate Neurotoxicity in Primary Cortical Culturesxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88:6368-71 (1991); and Dawson et al., xe2x80x9cMechanisms of Nitric Oxide-mediated Neurotoxicity in Primary Brain Culturesxe2x80x9d, J. Neurosci., 13:6, 2651-61 (1993), Dawson et al., xe2x80x9cResistance to Neurotoxicity in Cortical Cultures from Neuronal Nitric Oxide Synthase-Deficient Micexe2x80x9d, J. Neurosci., 16:8, 2479-87 (1996), ladecola, xe2x80x9cBright and Dark Sides of Nitric Oxide in Ischemic Brain Injuryxe2x80x9d, Trends Neurosci., 20:3, 132-39 (1997), Huang et al., xe2x80x9cEffects of Cerebral Ischemia in Mice Deficient in Neuronal Nitric Oxide Synthasexe2x80x9d, Science, 265:1883-85 (1994), Beckman et al., xe2x80x9cPathological Implications of Nitric Oxide, Superoxide and Peroxynitrite Formationxe2x80x9d, Biochem. Soc. Trans., 21:330-34 (1993), and Szabxc3x3 et al., xe2x80x9cDNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, and Cellular Energy Depletion are Involved in the Cytotoxicity in Macrophages and Smooth Muscle Cells Exposed to Peroxynitritexe2x80x9d, Proc. Natl. Acad. Sci. USA, 93:1753-58 (1996).
It is also known that PARP inhibitors, such as 3-amino benzamide, affect DNA repair generally in response, for example, to hydrogen peroxide or gamma-radiation. Cristovao et al., xe2x80x9cEffect of a Poly(ADP-Ribose) Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced by Hydrogen Peroxide and xcex3-Radiation,xe2x80x9d Terato., Carcino., and Muta., 16:219-27 (1996). Specifically, Cristovao et al. observed a PARP-dependent recovery of DNA strand breaks in leukocytes treated with hydrogen peroxide.
PARP inhibitors have been reported to be effective in radiosensitizing hypoxic tumor cells and effective in preventing tumor cells from recovering from potentially lethal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair. U.S. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653.
Evidence also exists that PARP inhibitors are useful for treating inflammatory bowel disorders, such as colitis. Salzman et al., xe2x80x9cRole of Peroxynitrite and Poly(ADP-Ribose)Synthase Activation Experimental Colitis,xe2x80x9d Japanese J. Pharm., 75, Supp. I:15 (1997). Specifically, Colitis was induced in rats by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. Treated rats received 3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition of PARP activity reduced the inflammatory response and restored the morphology and the energetic status of the distal colon. See also, Southan et al., xe2x80x9cSpontaneous Rearrangement of Aminoalkylithioureas into Mercaptoalkylguanidines, a Novel Class of Nitric Oxide Synthase Inhibitors with Selectivity Towards the Inducible Isoformxe2x80x9d, Br. J. Pharm., 117:619-32 (1996); and Szabxc3x3 et al., xe2x80x9cMercaptoethylguanidine and Guanidine Inhibitors of Nitric Oxide Synthase React with Peroxynitrite and Protect Against Peroxynitrite-induced Oxidative Damagexe2x80x9d, J. Biol. Chem., 272:9030-36 (1997).
Evidence also exists that PARP inhibitors are useful for treating arthritis. Szabxc3x3 et al., xe2x80x9cProtective Effects of an Inhibitor of Poly(ADP-Ribose)Synthetase in Collagen-Induced Arthritis,xe2x80x9d Japanese J. Pharm., 75, Supp. I:102 (1997); Szabxc3x3 et al., xe2x80x9cDNA Strand Breakage, Activation of Poly(ADP-Ribose)Synthetase, and Cellular Energy Depletion are Involved in the Cytotoxicity in Macrophages and Smooth Muscle Cells Exposed to Peroxynitrite,xe2x80x9d Proc. Natl. Acad. Sci. USA, 93:1753-58 (March 1996); and Bauer et al., xe2x80x9cModification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-transformed Bovine Endothelial Cell Line by Treatment with 5-Iodo-6-amino-1,2-benzopyrone (INH2BP)xe2x80x9d, Intl. J. Oncol., 8:239-52 (1996); and Hughes et al., xe2x80x9cInduction of T Helper Cell Hyporesponsiveness in an Experimental Model of Autoimmunity by Using Nonmitogenic Anti-CD3 Monoclonal Antibodyxe2x80x9d, J. Immuno., 153:3319-25 (1994).
Further, PARP inhibitors appear to be useful for treating diabetes. Heller et al., xe2x80x9cInactivation of the Poly(ADP-Ribose)Polymerase Gene Affects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells,xe2x80x9d J. Biol. Chem., 270:19, 11176-80 (May 1995). Heller et al. used cells from mice with inactivated PARP genes and found that these mutant cells did not show NAD+ depletion after exposure to DNA-damaging radicals. The mutant cells were also found to be more resistant to the toxicity of NO.
PARP inhibitors have been shown to be useful for treating endotoxic shock or septic shock. Zingarelli et al., xe2x80x9cProtective Effects of Nicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failure in Endotoxic Shock: Potential Involvement of PolyADP Ribosyl Synthetase,xe2x80x9d Shock, 5:258-64 (1996), suggests that inhibition of the DNA repair cycle triggered by poly(ADP ribose) synthetase has protective effects against vascular failure in endotoxic shock. Zingarelli et al. found that nicotinamide protects against delayed, NO-mediated vascular failure in endotoxic shock. Zingarelli et al. also found that the actions of nicotinamide may be related to inhibition of the NO-mediated activation of the energy-consuming DNA repair cycle, triggered by poly(ADP ribose) synthetase. Cuzzocrea, xe2x80x9cRole of Peroxynitrite and Activation of Poly(ADP-Ribose) Synthetase in the Vascular Failure Induced by Zymosan-activated Plasma,xe2x80x9d Brit. J. Pharm., 122:493-503 (1997).
PARP inhibitors have been used to treat cancer. Suto et al., xe2x80x9cDihydroisoquinolinones: The Design and Synthesis of a New Series of Potent Inhibitors of Poly(ADP-Ribose) Polymerasexe2x80x9d, Anticancer Drug Des., 7:107-17 (1991). In addition, Suto et al., U.S. Pat. No. 5,177,075, discusses several isoquinolines used for enhancing the lethal effects of ionizing radiation or chemotherapeutic agents on tumor cells. Weltin et al., xe2x80x9cEffect of 6(5H)-Phenanthridinone, an Inhibitor of Poly(ADP-ribose) Polymerase, on Cultured Tumor Cellsxe2x80x9d, Oncol. Res., 6:9, 399-403 (1994), discusses the inhibition of PARP activity, reduced proliferation of tumor cells, and a marked synergistic effect when tumor cells are co-treated with an alkylating drug.
Still another use for PARP inhibitors is the treatment of peripheral nerve injuries, and the resultant pathological pain syndrome known as neuropathic pain, such as that induced by chronic constriction injury (CCI) of the common sciatic nerve and in which transsynaptic alteration of spinal cord dorsal horn characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called xe2x80x9cdarkxe2x80x9d neurons) occurs. Mao et al., Pain, 72:355-366 (1997).
PARP inhibitors have also been used to extend the lifespan and proliferative capacity of cells including treatment of diseases such as skin aging, Alzheimer""s disease, atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseases of skeletal muscle involving replicative senescence, age-related muscular degeneration, immune senescence, AIDS, and other immune senescence diseases; and to alter gene expression of senescent cells. WO 98/27975.
Large numbers of known PARP inhibitors have been described in Banasik et al., xe2x80x9cSpecific Inhibitors of Poly(ADP-Ribose) Synthetase and Mono(ADP-Ribosyl)-Transferasexe2x80x9d, J. Biol. Chem., 267:3, 1569-75 (1992), and in Banasik et al., xe2x80x9cInhibitors and Activators of ADP-Ribosylation Reactionsxe2x80x9d, Molec. Cell. Biochem., 138:185-97 (1994). However, effective use of these PARP inhibitors, in the ways discussed above, has been limited by the concurrent production of unwanted side-effects (Milam et al., xe2x80x9cInhibitors of Poly(Adenosine Dipbosphate-Ribose) Synthesis: Effect on Other Metabolic Processesxe2x80x9d, Science, 223:589-91 (1984)).
There continues to be a need for effective and potent PARP inhibitors which produce minimal side-effects. The present invention provides compounds, compositions for, and methods of, inhibiting PARP activity for treating and/or preventing cellular, tissue and/or organ damage resulting from cell damage or death due to, for example, necrosis or apoptosis. The compounds and compositions of the present invention are specifically useful in ameliorating, treating and/or preventing neural tissue or cell damage, including that following focal ischemia and reperfusion injury. Generally, inhibition of PARP activity spares the cell from energy loss, preventing irreversible depolarization of the neurons and, thus, provides neuroprotection. While not wishing to be bound by any mechanistic theory, the inhibition of PARP activity by use of the compounds, compositions and methods of the present invention is believed to protect cells, tissue and organs by protection against the ill-effects of reactive free radicals and nitric oxide. The present invention therefore also provides methods of treating and/or preventing cells, tissue and/or organs from reactive free radical and/or nitric oxide induced damage or injury.
The present invention provides 6(5H)phenan- or (5H)phenanthridin-6-one compounds which inhibit poly(ADP-ribose) polymerase (xe2x80x9cPARPxe2x80x9d), compositions containing these compounds and methods for making and using these PARP inhibitors to treat, prevent and/or ameliorate the effects of the conditions described herein.
The compounds of the present invention are broadly described by the following Formula I: 
or a pharmaceutically acceptable salt, hydrate, prodrug, or mixtures thereof, wherein:
R1 hydrogen or halogen;
R2 is hydrogen, hydroxyl, amino, nitroso, methyl, amino methyl or carboxylic acid, preferably R1 and R2 are not both hydrogen;
one of R3 and R4 is xe2x80x94QP and the other of R3 and R4 is one of hydrogen, methyl, trifluoromethyl, nitro, amino, halogen and 1-piperazine,
wherein Q is one of 
P is Zxe2x80x94(N(R5R6)), such as Zxe2x80x94N(R5R6), Z or a 5 or 6 membered substituted or unsubstituted aromatic or non-aromatic ring which contains 0, 1, 2 or 3 heteroatoms selected from the group consisting of O, N, S and a combination of two or three of O, N and S;.
wherein A is carbon or Sxe2x95x90O,
X is O, S , N or an N-substituted amino acid; provided that,
when X is O or S, then Y is absent,
when X is N, then Y is hydrogen, C1-C6 straight or branched chain alkyl, optionally substituted alkoxy or alkyl amino, or Y and Z are taken together to form a 5, 6 or 7 membered substituted or unsubstituted heterocyclic aromatic or non-aromatic ring which contains 1, 2 or 3 heteroatoms selected from O, N, S and mixtures combination;
Z is hydrogen, a direct bond, a carbonyl, an optionally substituted C1-C5 straight or branched chain alkyl, cycloalkyl, carboxy, optionally substituted C1-C6 ether, aryl or heteroaryl, alkylalkenyl, alkynyl, alkylhalo, straight or branched chain or CH2COOH, provided that when P is Z, Z is not hydrogen and
R5 and R6 are independently hydrogen, lower alkyl, lower alkenyl, lower alkanol, heterocycle, heteroaryl, alkoxy, aryloxy, alkylamino, arylamino, CH2COOH, or R5 and R6 taken together form a 5, 6 or 7 membered substituted or unsubstituted heterocyclic or cycloalkyl ring containing 1, 2 or 3 heteroatoms selected from O, N, S and combinations thereof.